Rapid, high-throughput, cost-effective whole-genome sequencing of SARS-CoV-2 using a condensed library preparation of the Illumina DNA Prep kit.

The ongoing COVID-19 pandemic necessitates cost-effective, high-throughput, and timely whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses for outbreak investigations, identifying variants of concern (VoC), characterizing vaccine breakthrough infections, and public health surveillance. In addition, the enormous demand for WGS on supply chains and the resulting shortages of laboratory supplies necessitated the use of low-reagent and low-consumable methods. Here, we report an optimized library preparation method (the BCCDC cutdown method) that can be used in a high-throughput scenario, where one technologist can perform 576 library preparations (6 plates of 96 samples) over the course of one 8-hour shift. The same protocol can also be used in a rapid turnaround time scenario, from primary samples (up to 96 samples) to loading on a sequencer in an 8-hour shift. This new method uses Freed et al.'s 1,200 bp primer sets (Biol Methods Protoc 5:bpaa014, 2020, https://doi.org/10.1093/biomethods/bpaa014) and a modified and condensed Illumina DNA Prep workflow (Illumina, CA, USA). Compared to the original protocol, the application of this new method using hundreds of clinical specimens demonstrated equivalent results to the full-length DNA Prep workflow at 45% of the cost, 15% of consumables required (such as pipet tips), 25% of manual hands-on time, and 15% of on-instrument time if performing on a liquid handler, with no compromise in sequence quality. Results demonstrate that this new method is a rapid, simple, cost-effective, and high-quality SARS-CoV-2 WGS protocol. IMPORTANCE: Sequencing has played an invaluable role in the response to the COVID-19 pandemic. Ongoing work in this area, however, demands optimization of laboratory workflow to increase sequencing capacity, improve turnaround time, and reduce cost without compromising sequence quality. This report describes an optimized DNA library preparation method for improved whole-genome sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen. The workflow advantages summarized here include significant time, cost, and consumable savings, which suggest that this new method is an efficient, scalable, and pragmatic alternative for SARS-CoV-2 whole-genome sequencing.

Targeted genomic sequencing of avian influenza viruses in wetland sediment from wild bird habitats.

Diverse influenza A viruses (IAVs) circulate in wild birds, including highly pathogenic strains that infect poultry and humans. Consequently, surveillance of IAVs in wild birds is a cornerstone of agricultural biosecurity and pandemic preparedness. Surveillance is traditionally done by testing wild birds directly, but obtaining these specimens is labor intensive, detection rates can be low, and sampling is often biased toward certain avian species. As a result, local incursions of dangerous IAVs are rarely detected before outbreaks begin. Testing environmental specimens from wild bird habitats has been proposed as an alternative surveillance strategy. These specimens are thought to contain diverse IAVs deposited by a broad range of avian hosts, including species that are not typically sampled by surveillance programs. To enable this surveillance strategy, we developed a targeted genomic sequencing method for characterizing IAVs in these challenging environmental specimens. It combines custom hybridization probes, unique molecular index-based library construction, and purpose-built bioinformatic tools, allowing IAV genomic material to be enriched and analyzed with single-fragment resolution. We demonstrated our method on 90 sediment specimens from wetlands around Vancouver, Canada. We recovered 2,312 IAV genome fragments originating from all eight IAV genome segments. Eleven hemagglutinin subtypes and nine neuraminidase subtypes were detected, including H5, the current global surveillance priority. Our results demonstrate that targeted genomic sequencing of environmental specimens from wild bird habitats could become a valuable complement to avian influenza surveillance programs.IMPORTANCEIn this study, we developed genome sequencing tools for characterizing avian influenza viruses in sediment from wild bird habitats. These tools enable an environment-based approach to avian influenza surveillance. This could improve early detection of dangerous strains in local wild birds, allowing poultry producers to better protect their flocks and prevent human exposures to potential pandemic threats. Furthermore, we purposefully developed these methods to contend with viral genomic material that is diluted, fragmented, incomplete, and derived from multiple strains and hosts. These challenges are common to many environmental specimens, making these methods broadly applicable for genomic pathogen surveillance in diverse contexts.

Targeted genomic sequencing with probe capture for discovery and surveillance of coronaviruses in bats.

Public health emergencies like SARS, MERS, and COVID-19 have prioritized surveillance of zoonotic coronaviruses, resulting in extensive genomic characterization of coronavirus diversity in bats. Sequencing viral genomes directly from animal specimens remains a laboratory challenge, however, and most bat coronaviruses have been characterized solely by PCR amplification of small regions from the best-conserved gene. This has resulted in limited phylogenetic resolution and left viral genetic factors relevant to threat assessment undescribed. In this study, we evaluated whether a technique called hybridization probe capture can achieve more extensive genome recovery from surveillance specimens. Using a custom panel of 20,000 probes, we captured and sequenced coronavirus genomic material in 21 swab specimens collected from bats in the Democratic Republic of the Congo. For 15 of these specimens, probe capture recovered more genome sequence than had been previously generated with standard amplicon sequencing protocols, providing a median 6.1-fold improvement (ranging up to 69.1-fold). Probe capture data also identified five novel alpha- and betacoronaviruses in these specimens, and their full genomes were recovered with additional deep sequencing. Based on these experiences, we discuss how probe capture could be effectively operationalized alongside other sequencing technologies for high-throughput, genomics-based discovery and surveillance of bat coronaviruses.

ProbeTools: designing hybridization probes for targeted genomic sequencing of diverse and hypervariable viral taxa.

BACKGROUND: Sequencing viruses in many specimens is hindered by excessive background material from hosts, microbiota, and environmental organisms. Consequently, enrichment of target genomic material is necessary for practical high-throughput viral genome sequencing. Hybridization probes are widely used for enrichment in many fields, but their application to viral sequencing faces a major obstacle: it is difficult to design panels of probe oligo sequences that broadly target many viral taxa due to their rapid evolution, extensive diversity, and genetic hypervariability. To address this challenge, we created ProbeTools, a package of bioinformatic tools for generating effective viral capture panels, and for assessing coverage of target sequences by probe panel designs in silico. In this study, we validated ProbeTools by designing a panel of 3600 probes for subtyping the hypervariable haemagglutinin (HA) and neuraminidase (NA) genome segments of avian-origin influenza A viruses (AIVs). Using in silico assessment of AIV reference sequences and in vitro capture on egg-cultured viral isolates, we demonstrated effective performance by our custom AIV panel and ProbeTools' suitability for challenging viral probe design applications. RESULTS: Based on ProbeTool's in silico analysis, our panel provided broadly inclusive coverage of 14,772 HA and 11,967 NA reference sequences. For each reference sequence, we calculated the percentage of nucleotide positions covered by our panel in silico; 90% of HA and NA references sequences had at least 90.8 and 95.1% of their nucleotide positions covered respectively. We also observed effective in vitro capture on a representative collection of 23 egg-cultured AIVs that included isolates from wild birds, poultry, and humans and representatives from all HA and NA subtypes. Forty-two of forty-six HA and NA segments had over 98.3% of their nucleotide positions significantly enriched by our custom panel. These in vitro results were further used to validate ProbeTools' in silico coverage assessment algorithm; 89.2% of in silico predictions were concordant with in vitro results. CONCLUSIONS: ProbeTools generated an effective panel for subtyping AIVs that can be deployed for genomic surveillance, outbreak prevention, and pandemic preparedness. Effective probe design against hypervariable AIV targets also validated ProbeTools' design and coverage assessment algorithms, demonstrating their suitability for other challenging viral capture applications.

A novel multiplex electrochemiluminescent immunoassay for detection and quantification of anti-SARS-CoV-2 IgG and anti-seasonal endemic human coronavirus IgG.

BACKGROUND: Multiplex immunoassays capture a comprehensive profile of the humoral response against SARS-CoV-2 and human endemic coronaviruses. We validated a multiplex panel (V-PLEX Panel 2) from Meso Scale Diagnostics targeting antibodies against nine coronavirus antigens. Performance was compared against alternative single- and multi-antigen immunoassays. METHODS: Sera collected for clinical or public health testing from 2018 to 2020 (n = 135) were used to compare all tested platforms, and inter-test agreement was assessed by Cohen's kappa coefficient. Sample category (positive/negative) was assigned based on collection date relative to the index case in Canada, and SARS-CoV-2 PCR and serology results. 117 out of the 135 samples (31 positive, 86 negative) were assigned a category and were used to calculate sensitivity and specificity, with MSD's test results based upon manufacturer-set cut-offs. RESULTS: We observed SARS-CoV-2 target sensitivities of 100% and specificities >94% for all antigens (RBD, Nucleocapsid, Spike) in V-PLEX Panel 2. When targets were combined, we found a SARS-CoV-2 sensitivity of 100% and specificity of 98.8% with no difference in performance compared to clinical assays, and Cohen's kappa ranging from 0.798 to 0.945 compared to surface plasmon resonance imaging (SPRi). Quantitative measurements of antibodies against the Spike protein of endemic human coronaviruses were concordant with SPRi. CONCLUSION: Meso Scale Diagnostics' V-PLEX Coronavirus Panel 2 allows for highly sensitive and specific detection of anti-coronavirus IgG, and is concordant with other serological assays for detection of antibodies against SARS-CoV-2 and the endemic human coronaviruses, making it a good tool for humoral response characterization after both infection and vaccination.

Mutations in emerging variant of concern lineages disrupt genomic sequencing of SARS-CoV-2 clinical specimens.

Mutations in emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages can interfere with laboratory methods used to generate viral genome sequences for public health surveillance. We identified 20 mutations that are widespread in variant of concern lineages and affect widely used sequencing protocols by the ARTIC network and Freed et al. Three of these mutations disrupted sequencing of P.1 lineage specimens during a recent outbreak in British Columbia, Canada. We provide laboratory validation of protocol modifications that restored sequencing performance. The study findings indicate that genomic sequencing protocols require immediate updating to address emerging mutations. This work also suggests that routine monitoring and protocol updates will be necessary as SARS-CoV-2 continues to evolve. The bioinformatic and laboratory approaches used here provide guidance for this kind of assay maintenance.

The contrasting role of nasopharyngeal angiotensin converting enzyme 2 (ACE2) transcription in SARS-CoV-2 infection: A cross-sectional study of people tested for COVID-19 in British Columbia, Canada.

BACKGROUND: Angiotensin converting enzyme 2 (ACE2) protein serves as the host receptor for SARS-CoV-2, with a critical role in viral infection. We aim to understand population level variation of nasopharyngeal ACE2 transcription in people tested for COVID-19 and the relationship between ACE2 transcription and SARS-CoV-2 viral load, while adjusting for expression of: (i) the complementary protease, Transmembrane serine protease 2 (TMPRSS2), (ii) soluble ACE2, (iii) age, and (iv) biological sex. The ACE2 gene was targeted to measure expression of transmembrane and soluble transcripts. METHODS: A cross-sectional study of n = 424 "participants" aged 1-104 years referred for COVID-19 testing was performed in British Columbia, Canada. Patients who tested positive for COVID-19 were matched by age and biological sex to patients who tested negative. Viral load and host gene expression were assessed by quantitative reverse-transcriptase polymerase chain reaction. Bivariate analysis and multiple linear regression were performed to understand the role of nasopharyngeal ACE2 expression in SARS-CoV-2 infection. FINDINGS: Analysis showed no association between age and nasopharyngeal ACE2 transcription in those who tested negative for COVID-19 (P = 0•092). Mean relative transcription of transmembrane (P = 0•00012) and soluble (P<0•0001) ACE2 isoforms, as well as TMPRSS2 (P<0•0001) was higher in COVID-19-negative participants than COVID--19 positive ones, yielding a negative correlation between targeted host gene expression and positive COVID-19 diagnosis. In bivariate analysis of COVID-19-positive participants, transcription of transmembrane ACE2 positively correlated with SARS-CoV-2 viral RNA load (B = 0•49, R2=0•14, P<0•0001), transcription of soluble ACE2 negatively correlated (B= -0•85, R2= 0•26, P<0•0001), and no correlation was found with TMPRSS2 transcription (B= -0•042, R2=<0•10, P = 0•69). Multivariable analysis showed that the greatest viral RNA loads were observed in participants with high transmembrane ACE2 transcription (Β= 0•89, 95%CI: [0•59 to 1•18]), while transcription of the soluble isoform appears to protect against high viral RNA load in the upper respiratory tract (Β= -0•099, 95%CI: [-0•18 to -0•022]). INTERPRETATION: Nasopharyngeal ACE2 transcription plays a dual, contrasting role in SARS-CoV-2 infection of the upper respiratory tract. Transcription of the transmembrane ACE2 isoform positively correlates, while transcription of the soluble isoform negatively correlates with viral RNA load after adjusting for age, biological sex, and transcription of TMPRSS2. FUNDING: This project (COV-55) was funded by Genome British Columbia as part of their COVID-19 rapid response initiative.

Assessing oligonucleotide designs from early lab developed PCR diagnostic tests for SARS-CoV-2 using the PCR_strainer pipeline.

INTRODUCTION: During the first month of the SARS-CoV-2 outbreak, rapid development of PCR-based diagnostic tests became a global priority so that timely diagnosis, isolation, and contact tracing could minimize the advancing pandemic surge. Designing these tests for broad, long-term detection was complicated by limited information about the novel virus' genome sequence and how it might mutate during global spread and adaptation to humans. METHODS: We assessed eight widely adopted lab developed PCR tests for SARS-CoV-2 against 15,001 SARS-CoV-2 genome sequences. Using a custom bioinformatic pipeline called PCR_strainer, we identified all mismatches and sequence variants in genome locations targeted by 15 sets of primer/probe oligonucleotides from these assays. RESULTS: For 12 out of 15 primer/probe sets, over 98 % of SARS-CoV-2 genomes had no mismatches. Two primer/probe sets contained a single mismatch in the reverse primer that was present in over 99 % of genomes. One primer/probe set targeted a location with extensive polymorphisms with 23 sequence observed variants at the forward primer location. One of these variants, which contains three nucleotide mismatches, arose in February as part of the emergence of a viral clade and was present in 18.8 % of the genomes we analyzed. DISCUSSION: Most early PCR diagnostic tests for SARS-CoV-2 remain inclusive of circulating viral diversity, but three assays with extensive mismatches highlight assay design challenges for novel pathogens and provide valuable lessons for PCR assay design during future outbreaks. Our bioinformatics pipeline is also presented as a useful general-purpose tool for assessing PCR diagnostics assays against circulating strains.

Characterization of Legionella Species from Watersheds in British Columbia, Canada.

Legionella spp. present in some human-made water systems can cause Legionnaires' disease in susceptible individuals. Although legionellae have been isolated from the natural environment, variations in the organism's abundance over time and its relationship to aquatic microbiota are poorly understood. Here, we investigated the presence and diversity of legionellae through 16S rRNA gene amplicon and metagenomic sequencing of DNA from isolates collected from seven sites in three watersheds with varied land uses over a period of 1 year. Legionella spp. were found in all watersheds and sampling sites, comprising up to 2.1% of the bacterial community composition. The relative abundance of Legionella tended to be higher in pristine sites than in sites affected by agricultural activity. The relative abundance levels of Amoebozoa, some of which are natural hosts of legionellae, were similarly higher in pristine sites. Compared to other bacterial genera detected, Legionella had both the highest richness and highest alpha diversity. Our findings indicate that a highly diverse population of legionellae may be found in a variety of natural aquatic sources. Further characterization of these diverse natural populations of Legionella will help inform prevention and control efforts aimed at reducing the risk of Legionella colonization of built environments, which could ultimately decrease the risk of human disease. IMPORTANCE Many species of Legionella can cause Legionnaires' disease, a significant cause of bacterial pneumonia. Legionella in human-made water systems such as cooling towers and building plumbing systems are the primary sources of Legionnaires' disease outbreaks. In this temporal study of natural aquatic environments, Legionella relative abundance was shown to vary in watersheds associated with different land uses. Analysis of the Legionella sequences detected at these sites revealed highly diverse populations that included potentially novel Legionella species. These findings have important implications for understanding the ecology of Legionella and control measures for this pathogen that are aimed at reducing human disease.

A comprehensive method for amplicon-based and metagenomic characterization of viruses, bacteria, and eukaryotes in freshwater samples.

BACKGROUND: Studies of environmental microbiota typically target only specific groups of microorganisms, with most focusing on bacteria through taxonomic classification of 16S rRNA gene sequences. For a more holistic understanding of a microbiome, a strategy to characterize the viral, bacterial, and eukaryotic components is necessary. RESULTS: We developed a method for metagenomic and amplicon-based analysis of freshwater samples involving the concentration and size-based separation of eukaryotic, bacterial, and viral fractions. Next-generation sequencing and culture-independent approaches were used to describe and quantify microbial communities in watersheds with different land use in British Columbia. Deep amplicon sequencing was used to investigate the distribution of certain viruses (g23 and RdRp), bacteria (16S rRNA and cpn60), and eukaryotes (18S rRNA and ITS). Metagenomic sequencing was used to further characterize the gene content of the bacterial and viral fractions at both taxonomic and functional levels. CONCLUSION: This study provides a systematic approach to separate and characterize eukaryotic-, bacterial-, and viral-sized particles. Methodologies described in this research have been applied in temporal and spatial studies to study the impact of land use on watershed microbiomes in British Columbia.

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples.

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

Year-Long Metagenomic Study of River Microbiomes Across Land Use and Water Quality.

Select bacteria, such as Escherichia coli or coliforms, have been widely used as sentinels of low water quality; however, there are concerns regarding their predictive accuracy for the protection of human and environmental health. To develop improved monitoring systems, a greater understanding of bacterial community structure, function, and variability across time is required in the context of different pollution types, such as agricultural and urban contamination. Here, we present a year-long survey of free-living bacterial DNA collected from seven sites along rivers in three watersheds with varying land use in Southwestern Canada. This is the first study to examine the bacterial metagenome in flowing freshwater (lotic) environments over such a time span, providing an opportunity to describe bacterial community variability as a function of land use and environmental conditions. Characteristics of the metagenomic data, such as sequence composition and average genome size (AGS), vary with sampling site, environmental conditions, and water chemistry. For example, AGS was correlated with hours of daylight in the agricultural watershed and, across the agriculturally and urban-affected sites, k-mer composition clustering corresponded to nutrient concentrations. In addition to indicating a community shift, this change in AGS has implications in terms of the normalization strategies required, and considerations surrounding such strategies in general are discussed. When comparing abundances of gene functional groups between high- and low-quality water samples collected from an agricultural area, the latter had a higher abundance of nutrient metabolism and bacteriophage groups, possibly reflecting an increase in agricultural runoff. This work presents a valuable dataset representing a year of monthly sampling across watersheds and an analysis targeted at establishing a foundational understanding of how bacterial lotic communities vary across time and land use. The results provide important context for future studies, including further analyses of watershed ecosystem health, and the identification and development of biomarkers for improved water quality monitoring systems.

Identifying host sources, human health risk and indicators of Cryptosporidium and Giardia in a Canadian watershed influenced by urban and rural activities.

Cryptosporidium and Giardia were characterized in a watershed in southern Ontario, Canada, over a 2½ year period. River samples were collected every two weeks, primarily near a municipal drinking water treatment plant intake. Cryptosporidium and Giardia were frequently detected with an overall occurrence rate of 88 and 97%, respectively. Giardia concentrations were higher than Cryptosporidium, with median values of 80 cysts 100 L(-1) and 12 oocysts 100 L(-1), respectively. Although pathogens rarely show a significant relationship with fecal or water quality indicators, this study determined that Cryptosporidium, but not Giardia, was significantly correlated with Escherichia coli, turbidity and river flow. There was no correlation between the two types of protozoa, and only Giardia showed a seasonal trend with higher concentrations at cold water temperatures. Cryptosporidium genotyping of all samples found that farm animals and wildlife were an important contributor of oocysts in the watershed, and that Cryptosporidium strains/genotypes of medium to high risk for human infection (C. hominis, C. parvum and C. ubiquitum) were detected in 16% of samples. This study was able to identify Cryptosporidium host sources and human health risk, and to identify differences between Cryptosporidium and Giardia occurrence in the watershed.